This invention relates to methods for detecting 5T4 RNA in bodily fluids including but not limited to plasma and serum Ribonucleic acid (RNA) is essential to the processes that allow translation of the genetic code to form proteins necessary for cellular functions, both in normal and neoplastic cells. While the genetic code structurally exists as deoxyribonucleic acid (DNA), it is the function of RNA to carry and translate this code to the cellular sites of protein production. The pathogenesis and regulation of cancer is dependent upon RNA-mediated translation of specific genetic codes to produce proteins involved with cell proliferation, regulation, and death, including but not limited to those RNA associated with specific cellular processes characteristic of cancer, such as processes associated with metastatic potential, invasiveness, and alterations of cell-cell interactions. Furthermore, some RNA and their translated proteins, although not necessarily involved in specific neoplastic pathogenesis or regulation, may serve to delineate recognizable characteristics of particular neoplasms by either being elevated or inappropriately expressed. The RNA associated with cancer and premalignant or neoplastic states have been referred to herein as tumor-derived, or tumor-associated RNA. The invention, as described in U.S. patent application Ser. No. 09/155,152, incorporated by reference herein in its entirety, provides a method by which tumor-associated or tumor-derived RNA in bodily fluids such as plasma and serum can be detected and thus utilized for the detection, monitoring, or evaluation of cancer or premalignant conditions.
5T4 is a transmembrane glycoprotein present in trophoblast tissue whose gene structure has recently been characterized (Hole, 1988; Hole, 1990; Myers, 1994; King, 1999). The protein is only expressed at low levels on cells of a few other normal epithelium. Significantly, 5T4 expression is upregulated in the cells of many epithelial cancers and premalignant tissues, including but not limited to those of the breast, ovary, lung, cervix, colorectum, stomach, pancreas, bladder, endometrium, brain, kidney, and esophagus (Jones, 1990; Southall, 1990; Starzynska, 1992; Starzynska, 1994), and its mRNA is thereby a tumor-associated RNA. Overexpression of 5T4 is particularly associated with cancers of high metastatic potential and worse prognosis (Mulder, 1997; Styns; 1994). Detection of 5T4 thereby provides a method for detecting and monitoring a wide spectrum of cancers and premalignancies, and may have prognostic; significance. 5T4 further provides a potential target for cancer therapies, particularly monoclonal antibody-based therapies. 5T4 thus appear an important tumor marker, and a test of blood or other bodily fluids that detects the presence of 5T4 would be useful. However, the 5T4 protein has not been reported to be shed from the cell surface or to circulate in blood.
The present invention describes a method of evaluating for 5T4 by detecting 5T4 mRNA in blood, particularly plasma and serum, and other bodily fluids including but not limited to urine, effusions, ascites, saliva, cerebrospinal fluid, cervical, vaginal, and endometrial secretions, gastrointestinal secretions, bronchial secretions, and associated tissue washings.
The present invention provides a method for detecting of 5T4 RNA in blood or a blood fraction, including plasma and serum, and other bodily fluids, the method comprising the steps of extracting RNA from blood, plasma, serum, and other bodily fluid, amplifying 5T4 mRNA or its cDNA, and detecting the amplified product of 5T4 mRNA or its cDNA.
In a first aspect, the present invention provides methods for detecting 5T4 RNA in blood or blood fractions, including plasma and serum, in a human as an aid in the detection, diagnosis, monitoring, treatment, or evaluation of neoplastic disease, including early cancer, non-invasive cancer, carcinoma in-situ, premalignancy, invasive cancer; advanced cancer, and benign neoplasm, wherein the method comprises the steps of extracting RNA from blood or blood plasma or serum, amplifying a fraction of the extracted RNA or the corresponding cDNA wherein said fraction comprises 5T4 RNA, and detecting the amplified product of 5T4 RNA or its cDNA.
The invention further provides a method for detecting 5T4 RNA in all bodily fluids including but not limited to whole blood, plasma, serum, urine, effusions, ascitic fluid, saliva, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including sputum, secretions or washings from the breast, and other associated tissue washings from a human as an aid in the detection, diagnosis, monitoring, treatment, or evaluation of neoplastic disease, including early cancer, non-invasive cancer, carcinoma in-situ, premalignancy, invasive cancer, advanced cancer, and benign neoplasm, wherein the method comprises the steps of extracting RNA from the bodily fluid, amplifying a fraction of the extracted RNA or the corresponding cDNA wherein the fraction comprises 5T4 RNA, and detecting the amplified product of 5T4 RNA or its cDNA.
The invention thereby provides the method of amplifying and detecting extracellular 5T4 RNA.
The method of the invention further provides a convenient method of detecting 5T4 RNA in cells and tissue from a human, wherein the method comprises the steps of extracting RNA from cells or tissue, amplifying a fraction of the extracted RNA or the corresponding cDNA wherein said fraction comprises 5T4 RNA, and detecting the amplified product of 5T4 RNA or its cDNA.
The invention provides for primers useful in the amplification of 5T4 mRNA or its cDNA.
The invention provides for a diagnostic kit enabling detection of 5T4 RNA, in which primers or probes used in the amplification of 5T4 RNA or its cDNA are provided. In preferred embodiments of the inventive methods, 5T4 RNA is extracted from blood, plasma, serum, or other bodily fluids using an extraction method selected from a group consisting of gelatin extraction method; silica, glass bead, or diatom extraction method; guanidinium thiocyanate acid-phenol based extraction methods; guanidinium; thiocyanate acid based extraction methods; centrifugation through a cesium chloride or similar gradient; phenol-chloroform based extraction methods; or other commercially available RNA extraction methods.
In preferred embodiments of the inventive methods, 5T4 RNA or its cDNA is amplified using an amplification method selected from a group consisting of reverse transcriptase polymerase chain reaction; ligase chain reaction; DNA signal amplification; amplifiable RNA reporters; Q-beta replication; transcription-based amplification; isothermal nucleic acid sequence based amplification; self-sustained sequence replication assays; boomerang DNA amplification; strand displacement activation; cycling probe technology; and any combination or variation thereof.
In preferred embodiments of the inventive methods, detection of the amplified 5T4 RNA or 5T4 cDNA product is performed using a detection method selected from a group consisting of gel electrophoresis; ELISA detection including modifications, including biotinylated or otherwise modified primers; hybridization using a specific, fluorescent-, radioisotope-, or chromogenically-labeled probe; Southern blot analysis; electrochemiluminescence; reverse dot blot detection; and high-performance liquid chromatography.
In a particularly preferred embodiment, 5T4 RNA is reverse transcribed to its cDNA prior to amplification.
The methods of the invention are provided as diagnostic methods for detecting 5T4 RNA in a human at risk for developing or who has developed a neoplastic, premalignant, or malignant disease consisting of cells expressing 5T4 RNA, wherein the methods comprise the steps of extracting RNA from bodily fluid, amplifying a fraction of the extracted RNA or the corresponding cDNA wherein said fraction comprises 5T4 RNA, and detecting the amplified product.
The methods of the invention thereby particularly provide diagnostic methods for identifying humans at risk for developing or who have malignancy or premalignancy of the epithelium, these malignancies including but not limited to breast, ovarian, lung, cervical, colorectal, gastric, pancreatic, bladder, endometrial, brain, kidney, and esophageal cancers, and these premalignancies and carcinoma in-situ including but not limited to cervical dysplasia, cervical intraepithelial neoplasia (CIN), bronchial dysplasia, atypical hyperplasia of the breast, ductal carcinoma in-situ, colorectal adenoma, atypical endometrial hyperplasia, and Barrett""s esophagus.
The methods of the invention further provide a method to identify or select a human having 5T4 expressing malignancy or premalignancy. The invention thereby provides a method to identify, stratify, or select a human who might benefit from a 5T4-directed therapy, or from a further diagnostic test.
It is therefore the object of this invention to detect or infer the presence of 5T4-positive cancerous or precancerous cells within a human having a recognized cancer or pre-cancer, and in those not previously diagnosed, by examining the plasma or serum fraction of blood, or examining other bodily fluid, for 5T4 mRNA in either a qualitative or quantitative fashion.
An advantageous application of this invention is to therefore allow identification of humans having epithelial malignancies and premalignancies.
Another advantageous application of this invention is to allow identification of humans having 5T4 expressing neoplasms.
Another advantageous application of this invention is to allow selection of humans for 5T4 directed therapies, including biotherapies such as monoclonal antibody therapy, anti-sense therapies, and vaccines.
Another advantageous application of this invention is to provide a marker as a guide to whether adequate therapeutic effect has been achieved, or whether additional or more advanced therapy is required, and to assess prognosis in these patients.
Another advantageous application of this invention is to allow identification or analysis, either quantitatively or quantitatively, of 5T4 RNA in plasma or serum of humans during or following surgical procedures to remove premalignant or malignant lesions, and thus allow stratification of such patients as to their risk of residual cancer following the surgery, and their need for further therapy.
Another advantageous application of this invention is to allow identification or analysis of 5T4 RNA, either qualitatively or quantitatively, in the blood or other bodily fluid of a human who has completed therapy as an early indicator or relapsed cancer, impending relapse, or treatment therapy.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.